Cells (Jun 2024)

A Reliable System for Quantitative G-Protein Activation Imaging in Cancer Cells

  • Elena Mandrou,
  • Peter A. Thomason,
  • Peggy I. Paschke,
  • Nikki R. Paul,
  • Luke Tweedy,
  • Robert H. Insall

DOI
https://doi.org/10.3390/cells13131114
Journal volume & issue
Vol. 13, no. 13
p. 1114

Abstract

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Fluorescence resonance energy transfer (FRET) biosensors have proven to be an indispensable tool in cell biology and, more specifically, in the study of G-protein signalling. The best method of measuring the activation status or FRET state of a biosensor is often fluorescence lifetime imaging microscopy (FLIM), as it does away with many disadvantages inherent to fluorescence intensity-based methods and is easily quantitated. Despite the significant potential, there is a lack of reliable FLIM-FRET biosensors, and the data processing and analysis workflows reported previously face reproducibility challenges. Here, we established a system in live primary mouse pancreatic ductal adenocarcinoma cells, where we can detect the activation of an mNeonGreen-Gαi3-mCherry-Gγ2 biosensor through the lysophosphatidic acid receptor (LPAR) with 2-photon time-correlated single-photon counting (TCSPC) FLIM. This combination gave a superior signal to the commonly used mTurquoise2-mVenus G-protein biosensor. This system has potential as a platform for drug screening, or to answer basic cell biology questions in the field of G-protein signalling.

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